This invention relates to the detection of DNA and RNA. In particular, this invention relates to the detection of DNA and mRNA expression levels using rolling circle amplification.
Rolling circle amplification (RCA) is a nucleic amplification technique used with a xe2x80x98padlockxe2x80x99 oligonucleotide probe to detect single base changes in isolated nucleic acids (1-5). (Full citations for the references numerically identified herein are given before the claims.) While RCA is a powerful technique in theory, in practice it suffers from sensitivity and reproducibility problems.
Application of RCA to in situ targets in fixed or permeabilized cells has not been uniformly successful to date. Whereas recent work has demonstrated that the concept is viable (8), DNA detection efficiencies of 20-30% lessen the utility of RCA as an assay. Lack of success has been attributed to possible blocking of the polymerase by the target strand, and it was suggested that this problem might be overcome by cutting the target DNA strand near the RCA probe""s hybridization site (5). Under these conditions, DNA polymerase could free the probe from the target, in effect spinning the probe away from the target, keeping the polymerase from being blocked during the amplification process. However, this technique did not provide satisfactory results. Numerous attempts to achieve consistent in situ RCA have been made, mostly centered on the development of polymerases that can overcome the problems of stearic hindrance resulting from the RCA procedure. None have been successful; a pair of articles in Nature Genetics [2, 3] describes the necessity for and several possible solutions to in situ RCA, but neither demonstrates the process.
There is thus a need for improved RCA techniques. In particular, there is a need for improved in situ RCA techniques.
In order to meet these needs, the present invention is directed to improved RCA techniques for the detection of DNA and RNA. In particular, the present invention is directed to improved RCA in situ techniques for the detection of DNA and RNA.
In particular, the present invention is directed to the finding that in addition to restriction enzyme digestion of DNA, additional steps are required to achieve consistent and satisfactory results for RCA detection of DNA, particularly in situ DNA detection. Whereas heat denaturation is typically used to render the target DNA single stranded, the present invention is directed to the finding that complete removal of the non-targeted DNA strand by digestion with exonuclease significantly increases the efficiency of the RCA DNA detection process both in solution and in situ.
The present invention is also directed to the use of RCA to detect mRNA in situ. Using appropriate image analysis techniques, the RCA assay is sufficiently quantitative to enable transcriptionally-mediated dose-response curves to be generated.
Rolling circle amplification (RCA) is a versatile technology used to locate single-base substitutions in DNA and RNA which has proven to be very useful in detecting point mutations in extracellular nucleic acid but has not as yet been successful at detecting base changes in situ. This invention is also directed to a method of employing RCA in situ to detect gene copy number, single base mutations and gene expression levels in individual cultured cells and in tissue sections. By pre-treating DNA with a restriction enzyme and an exonuclease, one strand of the DNA helix is removed. This produces a single-stranded nucleic acid template in which minimal DNA-induced stearic hindrance exists to inhibit the polymerase activity necessary for RCA detection of DNA. The present invention is directed to the use of RCA to detect simultaneously single base changes in genomic DNA and levels of gene expression by amplifying transcribed RNA as well as DNA. When combined with gene isolation and sequencing technologies, the present invention can be used to detect and track mutations in cancer sections, enabling the course of genetic progression to be studied in situ.
The present invention is directed to a method of rolling circle amplification of DNA by: a) providing DNA; b) digesting the DNA with an endonuclease to form nicked DNA; c) digesting the nicked DNA with an exonuclease to prepare the DNA for rolling circle amplification and d) performing rolling circle amplification on the DNA. Such methods can be performed in solution and in situ.
The present invention is further directed to a method of preparing DNA for rolling circle amplification in situ, by a) fixing cells on a surface wherein the cells include DNA; b) digesting the DNA on the surface with an endonuclease to form nicked DNA; and c) digesting the nicked DNA with an exonuclease to prepare the DNA for rolling circle amplification.
In one format, the invention is directed to a method of performing rolling circle amplification in situ, by a) fixing cells on a surface wherein the cells include DNA; b) digesting the DNA on the surface with an endonuclease to form nicked DNA; c) digesting the nicked DNA with an exonuclease to form target DNA; d) ligating a padlock oligonucleotide probe to the target DNA to form ligated DNA and d) performing rolling circle amplification in situ on the ligated DNA.
In another format, the present invention is directed to a method of performing rolling circle amplification in situ, including: a) providing cells embedded in paraffin wherein the cells contain DNA; b) digesting the DNA in the paraffin with an endonuclease to form nicked DNA; c) digesting the nicked DNA with an exonuclease to form target DNA; d) ligating a padlock oligonucleotide to the target DNA to form ligated DNA; and e) performing rolling circle amplification in situ on the ligated DNA.
In one embodiment, the endonuclease is a restriction endonuclease and. Restriction endonucleases and exonuclease are commercially available from companies such as Promega Corporation, Madison Wis.
In another embodiment, the surface is a microscope slide coverslip or a microscope slide.
In another embodiment, the cells utilized in the method of the invention may be prokaryotic, eukaryotic plants or fungi. The eukaryotic cells may be mammalian including human, reptile, amphibian, avian or plant cells. The prokaryotic cells may be bacterial cells.
In another embodiment, the DNA may be selected from eukaryotic, prokaryotic, viral, chromomosomal, mitochondrial or chloroplast DNA.
In another format, the present invention is directed to a kit for rolling circle amplification, including: a) an exonuclease and b) an endonuclease. The kit may further include RCA reaction buffer. The kit may yet further include an oligonucleotide for RCA.
The present invention is further directed to a method of detecting RNA in situ, including: a) fixing cells on a surface wherein the cells include RNA; performing rolling circle amplification in situ on the RNA to detect the RNA in situ. In one format, the surface is a microscope slide. In another format, the cells are centrifuged onto the microscope slide
In another format, the cells are fixed by treatment with alcohol. The preferred alcohol is ethanol.
In another format, the invention is directed to a method for performing rolling circle amplification in situ to detect RNA, including: a) centrifuging cells onto a surface; b) fixing the cells on the surface with ethanol; c) hybridizing a padlock oligonucleotide probe to the RNA to form a DNA-RNA hybrid; d) ligating the oligonucleotide probe to the DNA of the DNA-RNA hybrid to form ligated DNA; e) performing rolling circle amplification on the ligated DNA to form amplified DNA; and f) detecting the amplified RNA by determining the levels of amplified DNA.